DNA Barcodes and Watermarks Study Leader: Steven M. Block Study Participants: David Donoho TerryHwa Gerald Joyce David Nelson Tim Stearns Peter Weinberger Ellen Williams June 2004 JSR-03-305 Approved for public release; distribution unlimited JASON The MITRE Corporation 7515 Colshire Drive McLean. Virginia 22102-7508 (703) 883-6997 Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of information estimaed to average 1 hour per response, including the time for review instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the cdlection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget. Paperwork Reduction Project (0704-0188), Washington, DC 20503. 1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED June 2004 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS DNA Barcodes and Watermarks 6. AUTHOR(S) 13049022-DC Steven Block, et al. 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER The MITRE Corporation JASON Program Office JSR-03-305 7515 Colshire Drive McLean, Virginia 22102 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/MONITORING AGENCY REPORT NUMBER Department of Energy Office of Science JSR-03-305 Washington, DC 20585 11. SUPPLEMENTARY NOTES 12a. DISTRIBUTION/AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE Approved for public release; distribution unlimited. Distribution Statement A 13. ABSTRACT (Maximum 200 words) This study was conducted on behalf of the DOE during the summer of 2003. The JASON Study explored the feasibility of a program to tag genetically the microorganisms used for bioremediation, for the purpose of identification. 14. SUBJECTTERMS 15. NUMBER OF PAGES 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT OF REPORT OFTHIS PAGE OF ABSTRACT SAR UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED Standard Form 298 (Rev. 2·89) Prescribed by ANSI Std. 239-18 298-102 Contents 1 EXECUTIVE SUMMARY ............................................................................................... 1 The Bioremediation Problem ............................................................................................... 1 The Barcode Concept. .......................................................................................................... 1 Barcode and Watermark Implementation ............................................................................ l Recommendations ................................................................................................................ 2 2 JASON STUDY (JSR-03-305) DNA BARCODES & WATERMARKS ...................... 5 Briefers We Heard ............................................................................................................... 5 Introduction: Statement of the Problem .............................................................................. 7 Multiple Types ofBioremediation ....................................................................................... 8 The DNA Barcode Concept ............................................................................................... 11 What a DNA Barcode is NOT ............................................................................................ 12 What a DNA Barcode IS .................................................................................................... 13 What is a DNA Watermark? .............................................................................................. 14 The BarcodelWatermark Tagging System ......................................................................... 15 Introduction to the Bacterial Chromosome ....................................................................... .15 Where to Place Barcodes & Watermarks .......................................................................... .16 Anatomy of a Barcode ....................................................................................................... 18 A Practical Barcode is Shown ............................................................................................ 20 How Many Practical Barcodes Exist? ............................................................................... 20 Constructing a DNA Watermark ....................................................................................... 22 Introducing Barcodes & Watermarks ................................................................................ 23 Placing Barcodes & Watermarks by Recommendation .................................................... .24 Placing Barcodes by Group II intron "Retro-homing" ...................................................... 25 Reading Barcodes & Watermarks ...................................................................................... 27 A Worked Example: Barcoding & Watermarking the Model Organism, D. radiodurans ............................ .29 Selecting the Barcode Insertion Site .................................................................................. 30 Choosing the Watermark Sites ........................................................................................... 31 An Alternative Barcode Scheme ........................................................................................ 32 A JASON Idea: Fast, Adjustable Molecular Clocks ......................................................... 33 A Clock Based on Tandem Repeats ................................................................................... 34 A Better Clock Based on a Directed, Error-Prone Polymerase ......................................... 35 Barcode and Watermark Considerations .......................................................................... .36 Barcodes and Bioremediation ............................................................................................ 37 JASON Recommendations .............................................................................................. 409 3 APPENDICES Appendix A: Construction and Analysis of DNA Barcode Libraries ............................ .43 Appendix B: Placing Barcodes and Watermarks by Recombination within the Genome .............................................................................. 50 1. EXECUTIVE SUMMARY The Bioremediation Problem It is estimated that 60% of DOE facilities contain groundwater contaminated by heavy metals, radionuclides, or chlorinated hydrocarbons. Moreover, 50% of the topsoil or sediment at DOE facilities is now contaminated. The DOE faces a massive remediation problem, with a need to treat 1.7 trillion gallons of polluted water and 40 million cubic meters of contaminated soil. To assist in this considerable effort, a "genomic approach to waste cleanup" is currently being explored, harnessing the power of microbial biochemistry (in communities of living bacteria and fungi) to degrade complex organic molecules, and to reduce or sequester certain chemicals, particularly heavy metals. Bioremediation can take on several forms, ranging from natural degradation ('intrinsic bioremediation'), to encouraging the growth of endogenous organisms in situ ('enhanced bioremediation'), to the introduction of non-native microbial species ('bioaugmentation'), to the application of sophisticated bioengineering to generate novel strains optimized for the specific remediation task at hand. Irrespective of the origin ofthe microbes used, however, it will be vital to establish a socially and legally acceptable means offollowing the growth and ecology ofall species usedfor bioremediation. The Barcode Concept This JASON Study explored the feasibility of a program to tag genetically the microorganisms used for bioremediation, for the purpose of identification. Such DNA-based tags would be fully heritable, but carefully designed to convey no phenotype to the organisms being labeled ("genotype without phenotype"). Tags would be structured so that they could be specifically amplified by PCR (using a universal set of primers) and rapidly read in the field or in the laboratory. The system we contemplate would support the sensitive, multiplexed readout of mixed populations of strains, such as those typically found in complex microbial communities. Moreover, DNA tags would be designed to be robust against most mutations and certain kinds of intentional interference. Properly implemented, these tags would constitute an effective 'barcode' system for tracking microorganisms in the wild. DNA barcode tracking could be used quantitatively to monitor microbial growth, dispersal, transport, blooms, die-outs, ecological niches, and more. All DNA barcode labels would be registered in a public database, and they would be designed and introduced following uniform standards established by the DOE. Barcode integrity could be further protected against tampering by a system of DNA 'watermarks'- a covert set of minor, distributed genomic changes - whose implementation details would remain proprietary. Barcode and Watermark Implementation A useful barcode system could be developed quite economically, adapting many of the methods currently available in genetics and molecular